PROJECT SUMMARY TB is now the number one infectious cause of death worldwide having recently surpassed HIV/AIDS. Despite its global importance, our understanding of the pathogen's fundamental strategies for evading human immune responses is limited, although it is now clear that the microbe utilizes an arsenal of molecular components to perturb phagolysosome maturation and autophagic killing. One recently recognized virulence strategy is that M.tb releases bacterial-derived nucleic acids (dsDNA and cyclic nucleotides such as cAMP and c-di-AMP) into the macrophage cytosol where they subvert innate immune signaling mediated by the host's cytosolic surveillance pathway (CSP). The central premise of this application is that in addition to dsDNA and cyclic nucleotides, M. tb RNA comprises yet another pathogen-derived molecule that gains access to the host cytosol and that M. tb RNA contributes to pathogenesis. Microbial RNA constitutes an important group of pathogen-associated molecular patterns (PAMPs) that potently stimulate innate immune activation. Although RNA of viral origin has been extensively studied as a trigger of the CSP, the concept that bacterial-derived RNA may be important in host pathogen interactions remains novel, and very little has been done to address the role of M.tb-derived RNA during infection. In exciting new preliminary data, we have observed that intracellular exposure to M.tb RNA skews host cell cytokine production. Additionally, we have discovered that loss of a key RNA sensor gene alters macrophage responses to M. tb infection. In this study we propose detailed exploratory studies to characterize the specific molecular determinants required for M.tb-derived RNA to trigger these responses. Additionally, we will use genome editing tools to generate targeted deletions of key pattern recognition receptors (PRRs) known to be RNA sensors in murine and human macrophages. These cell-lines will be evaluated for responsiveness to transfected M.tb RNA and for their ability to control intracellular M.tb proliferation. Further elucidation of the role of M.tb RNA in macrophage cell biology will better our understanding of M.tb pathogenesis and the microbe's ability to subvert innate immunity. More practically, these studies may also elucidate novel targets for pharmacologic and vaccine-based interventions to combat TB.